Specific Aims (Abstract) We will make fully operational a continuing pilot collaboration between the labs of Dr. Jessica P. Houston (NMSU) and Dr. Roger Brent (FHCRC). Our proposed work builds on complementary strengths in the two labs: instrumentation engineering and signal analysis in the Houston lab, protein engineering and genetics in the Brent lab. In particular, the work builds on frequency domain methods to determine fluorescence lifetimes in microscopy, and on signiflcantly more advanced developments in frequency domain methods for flow cytometry, for which Dr. Houston is emerging as a leader. Dysfunctions in cell signaling leading to inappropriate cell proliferation contribute to most cancers. Although much is known about cell signaling, key quesfions, including the causes and consequences of cellto- cell variation in signaling and response, remain unanswered. Here, we will use fluorescent lifefime methods to extend the power of single cell assays to quantify key events in cell signaling in yeast (the development platform) and in mammalian cells. These measurements depend on the use of Green Fluorescent Protein (GFP) derivatives fused to the proteins that make up the different cell signaling pathways to quantify events such as recruitment of a signaling complex to the cell membrane. During the next three years, we will develop our methods, to address these quesfions and to allow wider applicafions. In particular, we will confinue to improve our instrumentafion, use it to develop better GFP derivatives and signaling reporters, and use the improved methods to understand the causes of cell-to-cell variation in a yeast signaling system. We will also use these methods to develop high-signal reporters that allow quanfificafion of cell signaling in clonal, mammalian cell lines using flow cytometry. Successful work will merit future funding. It should also increase the visibility ofthe Houston lab and help recruit new students and researchers from underrepresented ethnic minorities, at both sites, into an active area of international scientific research.